Host determinants of enterovirus RNA replication and in vivo neuropathogenesis

项目摘要 病原体已经进化到利用细胞功能来支持它们的复制和传播， 先天免疫机制限制了它们的生长。细胞成分的发现和表征， 调节发病机制有望揭示治疗感染性疾病的新方法。肠道病毒 (EVs)包括大属的正极性单链RNA病毒，其成员引起 一些重要的人类疾病，如脊髓灰质炎、心肌炎、急性弛缓性麻痹和常见的 冷. EV如何利用细胞功能来促进复制并导致发病机制尚不完全 明白通过稳健的、无偏的敲除筛选方法，我们发现， 甲基转移酶SETD 3是广泛的人类EV感染所必需的。我们发现肠道病毒 在缺乏SETD 3的人类细胞中，复制受到严重阻碍，并且这种阻断发生在RNA聚合过程中。 复制步骤。SETD 3是一种甲基转移酶，可使肌动蛋白单甲基化，从而调节肌动蛋白功能。 然而，我们发现SETD 3的甲基转移酶活性不是其在病毒复制中的作用所必需的 这表明肠道病毒对SETD 3的依赖不依赖于肌动蛋白甲基化。我们进一步表明， SETD 3与几种肠道病毒的病毒非结构2A蛋白相互作用。SETD 3对于以下方面至关重要： 体内发病机制，因为我们表明Setd 3-/-小鼠完全免受致死性颅内接种 EV-A71在新生儿模型中。这些发现表明，SETD 3控制了一大类疾病的发病机制， 对人类健康具有强烈影响的病毒，包括可导致严重神经系统疾病的非脊髓灰质炎EV 症状（EV-A71，EV-D 68）。在本申请中，我们将确定SETD 3在病毒RNA中的特定作用， 复制，从结构上表征SETD 3和2A之间的相互作用，并测试假设， SETD 3与病毒非结构蛋白的相互作用是EV劫持的新分子机制 使基因组扩增成为可能的细胞机制。此外，为了研究SETD 3在小鼠中的体内作用， 模型，更忠实地概括了肠道肠道病毒的传播周期和发病机制，我们将 在免疫活性小鼠中建立并应用EV-A71口服感染模型。我们的结果将提供细节 关于宿主因子促进肠道病毒RNA复制的分子机制，揭示了非宿主因子是如何促进肠道病毒RNA复制的。 甲基转移酶的催化功能在微生物发病机制中起作用，并揭示了SETD 3在微生物发病机制中的体内作用。 促进EV-A71在参与初始复制、全身扩散和最终传播的不同细胞类型中的复制 在神经病理学中。

PROJECT SUMMARY Pathogens have evolved to co-opt cellular functions to support their replication and spread while inactivating innate immune mechanisms that restrict their growth. Discovery and characterization of cellular components that regulate pathogenesis hold promise for revealing new approaches to treat infectious diseases. Enteroviruses (EVs) comprise a large genus of single-stranded RNA viruses of positive polarity whose members cause a number of important human diseases such as poliomyelitis, myocarditis, acute flaccid paralysis and the common cold. How EVs co-opt cellular functions to promote replication and cause pathogenesis is incompletely understood. Through robust, unbiased knockout screening approaches, we have discovered that the protein methyltransferase SETD3 is required for infection by a broad range of human EVs. We showed that enterovirus replication is severely hampered in human cells lacking SETD3 and that the block occurs during the RNA replication step. SETD3 is a methyltransferase that mono-methylates actin, thereby regulating actin function. However, we found that methyltransferase activity of SETD3 is not required for its role in viral replication indicating that enteroviruses’ reliance on SETD3 is independent of actin methylation. We further showed that SETD3 interacts with the viral nonstructural 2A protein of several enteroviruses. SETD3 is critically important for in vivo pathogenesis as we show that Setd3-/- mice are completely protected from lethal intracranial inoculation with EV-A71 in a neonate model. These findings demonstrate that SETD3 controls pathogenesis for a large class of viruses with a strong impact on human health including non-polio EVs that can cause severe neurological symptoms (EV-A71, EV-D68). In this application, we will determine the specific role of SETD3 in viral RNA replication, structurally characterize the interaction between SETD3 and 2A, and test the hypothesis that SETD3’s interactions with viral nonstructural proteins are a novel molecular mechanism by which EVs hijack cellular machinery to enable genome amplification. Furthermore, to study the in vivo role of SETD3 in a mouse model that recapitulates more faithfully the transmission cycle and pathogenesis of enteric enteroviruses, we will develop and apply an oral infection model of EV-A71 in immune-competent mice. Our results will provide details on the molecular mechanisms by which host factors promote enteroviral RNA replication, reveal how non- catalytic functions of methyltransferases act in microbial pathogenesis and uncover the in vivo role of SETD3 in promoting EV-A71 replication in diverse cell types involved in initial replication, systemic spread and ultimately in neuropathogenesis.